Protein phosphorylation in nerve terminals: comparison of calcium/calmodulin-dependent and calcium/diacylglycerol-dependent systems.

Rat cerebral cortical synaptosomes that had been prelabeled with 32P-orthophosphate were exposed to either (1) K depolarization which causes Ca2+ influx and hence would be expected to activate Ca2+-dependent enzymes, including Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases (Ca/CaM kinases and protein kinase C, respectively); or (2) phorbol esters or 1-oleoyl-2-acetyl-glycerol (OAG), which selectively activate protein kinase C. Proteins whose state of phosphorylation was affected by these treatments could be divided into 3 classes. Class A includes 5 phosphoproteins that showed rapidly increased phosphorylation by synaptosomal depolarization but not by OAG or phorbol ester. Four of these proteins, synapsins Ia and Ib and proteins IIIa and IIIb, are neuron-specific, synaptic vesicle-associated proteins known to be substrates for Ca/CaM kinases I and II. These phosphoproteins were rapidly dephosphorylated upon synaptosomal repolarization. Class B is composed of 2 phosphoproteins that showed rapidly increased phosphorylation by either synaptosomal depolarization or treatment with phorbol ester or OAG. These 2 acidic proteins of Mr87 and 49 kDa are known from in vitro studies to be specific substrates for protein kinase C. Thermolytic peptide mapping indicated that the 87 kDa protein in synaptosomes was phosphorylated by protein kinase C in situ. These 2 phosphoproteins were slowly dephosphorylated upon synaptosomal repolarization. Class C comprises 4 phosphoproteins that were rapidly dephosphorylated upon synaptosomal depolarization and may be substrates for Ca2+-activated protein phosphatase(s). These data suggest that Ca2+ influx into nerve terminals activates Ca/CaM kinases I and II, protein kinase C, and unidentified protein phosphatase(s).(ABSTRACT TRUNCATED AT 250 WORDS)